IMMUNE RESPONSES IN VITRO : V. SUPPRESSION OFγM, γG,ANDγA PLAQUE-FORMING CELL RESPONSES IN CULTURES OF PRIMED MOUSE SPLEEN CELLS BY CLASS-SPECIFIC ANTIBODY TO MOUSE IMMUNOGLOBULINS

Suppression of Ig class-specific PFC responses by class-specific antibody to mouse immunoglobulin was studied in cultures of spleen cells from immunized mice. In contrast to cultures from normal mice where anti-µ suppressed responses in all Ig classes, anti-µ had progressively less suppressive effect on γ₁ and γ₂ responses in cultures from immunized mice with time after immunization. This was most pronounced at 10 days after immunization when anti-µ suppressed γM and γA responses, but had no or slight effect on γ₁ or γ₂ responses which were still suppressed with anti-γ₁ and anti-γ₂. These changes in precursor cell susceptibility to anti-µ were antigen specific.     

Immunological memory in mice. I. Physical separation and partial characterization of memory cells for different immunoglobulin classes from each other and from antibody-producing cells

Plaque forming cells (PFC) of different immunoglobulin classes producing antibodies against sheep erythrocytes were separated according to their buoyant densities by means of equilibrium centrifugation in a stepwise BSA gradient. In the period of 7–10 days after immunization γM PFC are markedly enriched in fractions of low density and relatively depleted in fractions of high density. The distribution of total γG PFC shows less enrichment in the lower density fractions and less depletion in the higher density fractions. The density profile for γG_2a PFC is even flatter, with a significant difference (depletion) relative to the unseparated spleen cells only in the highest density fraction. The density gradient distributions of cells able to transfer an adoptive immune response of the various immunoglobulin classes are markedly different from the PFC distribution. Cells obtained 7–10 days after immunization able to transfer an IgM response are present in the same proportions across the density gradient, whereas memory cells for γG_2a obtained at this time are markedly enriched in fractions of low density and virtually depleted from high density fractions. With increasing time after primary immunization, the γG_2a memory cells increase progressively in density and by 6 weeks the higher and lower density fractions have the same proportions of γG_2a memory cells. The total γG (mainly γG₁) memory cells by 7–10 days show slight enrichment in low density fractions and no depletion in high density fractions. The conclusions were reached that (a) memory for γG₁ develops earlier than memory for γG_2a and (b) that memory for anti-SRBC antibodies of different classes is carried in separate cells. When gradient fractions enriched for PFC and memory cells for all classes were completely depleted of PFC using glass bead columns, the ability of this fraction to transfer memory for all classes was not diminished. This shows that memory cells are not identical with cells secreting antibodies.     

IMMUNE RESPONSES IN VITRO : IV. SUPPRESSION OF PRIMARYγM, γG,ANDγA PLAQUE-FORMING CELL RESPONSES IN MOUSE SPLEEN CELL CULTURES BY CLASS-SPECIFIC ANTIBODY TO MOUSE IMMUNOGLOBULINS

The suppressive effects of monospecific goat anti-mouse globulins on primary immunoglobulin class-specific plaque-forming cell responses in mouse spleen cell cultures were investigated. Anti-µ suppressed responses in all immunoglobulin classes, whereas anti-γ₁ and anti-γ₂ suppressed the γ₁ and γ₂ responses but not γM or γA responses, and anti-γA suppressed only γA responses. The mechanism of action of the anti-µ was studied in detail because of its suppression of responses in all immunoglobulin classes. The anti-µ was specific for µ-chain determinants; its activity was dose dependent, but was not mediated by killing cells with surface µ-chain determinants. Free γM but not γG myeloma proteins in solution effectively competed with µ-bearing cells for the anti-µ. An excess of anti-µ was necessary in the cultures for 48 hr to insure complete suppression of 5-day responses. However, after removal of excess anti-µ at 48 hr, responses could be stimulated by newly added antigen in cultures where incubation was prolonged to 7 days. Anti-µ was most effective when added at the initiation of cultures and had no suppressive effect when added at 48 hr. Excess antigen did not effectively compete with anti-µ for antigen receptors. Precursors of antibody-forming cells were shown to be the cell population where the suppressive activity of anti-µ was mediated. The experiments suggest that anti-µ combines with µ-chain determinants in antigen-specific receptors on the surfaces of antibody-forming cell precursors, prevents effective stimulation by antigen and subsequent antibody production. To explain suppression of responses in all Ig classes by anti-µ, several models were proposed. It is not possible to determine from the data whether stimulation of precursor cells with γG or γA receptors requires concommitant stimulation of separate cells with only γM receptors, or whether cells bearing γM receptors are precommitted to or differentiate into cells capable of synthesis of other Ig classes, or whether receptors of γM and another Ig class are present on some virgin precursors or the second Ig receptor appears after antigenic stimulation.     

GM and INV factors in subclasses of human IgG

Human G myeloma (7S γ₂-myeloma) proteins were investigated for relationships between Gm and Inv genetic factors and the different antigenic types of heavy polypeptide chains (γ-chains) and light polypeptide chains. Myeloma proteins were isolated from the sera of 1 Chinese, 60 white and 28 Negro individuals. These 89 proteins were tested for eight Gm factors [Gm(a), Gm(x), Gm(b²), Gm(f), Gm(b¹), Gm(b³), Gm(b⁴), and Gm(c)], and two Inv factors [Inv(l) and Inv(b)]. Results of the tests were correlated with the four γ-chain subclasses (γ_2a, γ_2b, γ_2c and γ_2d) and the two types of light polypeptide chains, κ-chains (type K or I) and λ-chains (type L or II) found in human IgG molecules. 1. Gm factors were limited to myeloma proteins with heavy polypeptide chains of the γ_2b- and γ_2c-subclasses. No Gm factors were detected on γ_2a- and γ_2d-myeloma proteins or on a "heavy-chain" disease protein of subclass γ_2d. 2. γ_2b-Proteins were positive for at least one Gm factor and were either Gm(a+), Gm(a + x+), or Gm(b²+ f+). 3. γ_2c-Myeloma proteins, and one γ_2c-"heavy-chain" disease protein, were positive for at least one Gm factor and contained various combinations of factors Gm(b¹), (b²), (b⁴), and (c). Myeloma proteins from 3 Negroes were included in this group. 4. Inv factors (l) and (b) were limited to myeloma proteins with κ-light polypeptide chains. These Inv factors were not detected on proteins with λ-light polypeptide chains. 5. Most (70 per cent) of the γ_2b- and γ_2c-proteins with κ-chains were Inv(l+) or Inv(b+). None of the γ_2a- or γ_2d-proteins with κ-chains, however, contained these Inv factors.     

Immune responses in vitro. XI. Suppression of primary IgM and IgG plaque-forming cell responses in vitro by alloantisera against leukocyte alloantigens

The effects of alloantisera against leukocyte alloantigens on plaque-forming cell (PFC) responses to sheep erythrocytes and the terpolymer of L-glutamic acid⁶⁰-L-alanine³⁰-L-tyrosine¹⁰ (GAT) by mouse spleen cells in vitro have been investigated. Polyspecific antibodies against both H-2 and non-H-2 alloantigens on responding spleen cells suppressed both IgM and IgG PFC responses; antisera against alloantigens coded for by the K and I regions, but not the D region, of the H-2 complex also effectively suppressed PFC responses. The suppression was not due to cytotoxicity to the spleen cells or anti-immunoglobulin activity in the sera and was directly related to the amount of antiserum added to the cultures. The suppression was specific for spleen cells against which the alloantiserum was directed. The alloantisera suppressed responses most effectively when present during the first 24 h of incubation, and although not rendering lymphoid cells incapable of developing PFC responses after removal of noncell-bound antibody, did act by interfering with successful initiation of the PFC response. The alloantisera suppressed both IgM and IgG PFC responses when directed against alloantigens only on macrophages, but selectively suppressed IgG responses when directed against alloantigens only on lymphoid cells. The alloantisera did not interfere with the ability of macrophages to bind GAT or to support the viability of the lymphoid cells, but did interfere with the ability of macrophage-associated antigen to effectively stimulate antibody responses by the lymphoid cells. Possible mechanisms for the effects of alloantisera on macrophages and the selective suppression of IgG responses when the antisera are directed against alloantigens on lymphoid cells are discussed with reference to our current understanding of genetic restrictions governing cell interactions in the development of antibody responses in mice.     

Phosphodiesterase 4B in the cardiac L-type Ca²⁺ channel complex regulates Ca²⁺ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (I_Ca,L) is increased only in Pde4b^–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b^–/– and Pde4d^–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b^–/– mice but not in WT mice. These results identify PDE4B in the Ca_V1.2 complex as a critical regulator of I_Ca,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.     

Immunoglobulin isoantigens (allotypes) in the mouse. II. Allotypic analysis of three gammaG2-myeloma proteins from (NZB x BALB/c)F1 hybrids and of normal gammaG2-globulins

Further analysis of the isoantigens (allotypes) of 2 classes of normal mouse immunoglobulins, γG_2a and γG_2b, has shown a minimum of 10 specificities for the Ig-1 locus (controlling γG_2a-antigens) and 3 specificities for the Ig-3 locus (controlling γG_2b-antigens). Three γG₂-myeloma proteins of plasma cell tumors induced in (NZB x BALB/c)F₁ mice have been analyzed for the isoantigens they carry. NZB mice are genotypically Ig-1^e Ig-3^e, while BALB/c are Ig-1^a Ig-3^a. Two of the myeloma proteins are γG_2a-globulins. One of these, GPC-7, carries all the isoantigenic specificities of the Ig-1^e allele while the other, GPC-8, carries all the isoantigenic specificities of the Ig-1^a allele. Thus only one of the parental alleles of the mouse in which the tumor arose is expressed in each of these myeloma proteins. The third myeloma protein GPC-5, also carries the antigens of only one parental strain (NZB). However GPC-5, a γG_2b-globulin, carries only one of the Ig-3 specificities normally associated with γG_2b-globulins of NZB. Most remarkably it also carries one Ig-1 specificity normally associated with γG_2a-globulins of NZB. This is the first analyzed mouse myeloma shown (a) to express some but not all the antigenic specificities normally associated with an allele and (b) to carry antigenic specificities controlled by two distinct immunoglobulin loci. The implications of these findings are discussed in relation to the genetic control of immunoglobulins.     

Immunoglobulin and other surface antigens of cells of the immune system

Immunoglobulins (Ig) on cells of the immune system: The cytotoxicity test, with class-specific and type-specific anti-Ig sera, identifies κ and µ determinants on mouse lymphocytes. The proportion of κ⁺ cells is characteristic for each source of cells: 30% of bone marrow cells, 40% of cells from peripheral lymph nodes, 45% of lymphocytes from peripheral blood or peritoneal cavity, and 50% of spleen cells. No Ig was demonstrable on thymocytes or on leukemia cells (most of which arise from thymus-derived [T] cells). Cytotoxicity tests were performed on various myelomas secreting different Ig; the only positive reactions were given by κγ1 myelomas (all four κγ1 myelomas tested were sensitive to both anti-κ and anti-γ1). Hemolytic plaque-forming cells (PFC) of IgG type had no demonstrable surface Ig, but a proportion of IgM PFC were κ⁺µ⁺. Virtually all rosette-forming cells (RFC) have surface Ig, more than 90% of them being inhibited by anti-κ, 50% by anti-µ, and 10–30% by antisera to other heavy chains. Anti-λ sera gave no positive reactions with any cell type, which is in keeping with the low level of this light chain in mouse serum. Ig and other differentiation antigens as markers for T and B cells: Thymocytes are hallmarked by the alloantigens TL, θ, and the Ly series, and it is generally held that extrathymic lymphoid cells that bear them are derived from thymocytes. There is one alloantigen marker for the thymus-independent (B) cell, and that is PC, which appears late in differentiation. (The mouse-specific lymphocyte (MSLA) and mouse-specific bone marrow-derived lymphocyte (MBLA) antigens recognized by heteroantisera, not used in the present study, are other candidates for T and B cell markers.) Making use of antisera to these surface antigens to inhibit the function of cells that carry them, we find the following: Approximately 30% of RFC, 60% of IgM PFC, and 90% of IgG are PC⁺ and so are identified as B cells. No T markers were demonstrable on these cell populations. Thus if T cells do become RFC or PFC they presumably lose their T surface markers in the process (cf. the quantitative reduction of T markers accompanying the thymocyte → lymphocyte transition). Cells that have the potential to initiate graft-versus-host (GVH) reactions have the T cell surface phenotype θ⁺Ig^-. Adoptive transfer of thymus-dependent antibody-forming capacity (response to sheep erythrocytes) required θ⁺ cells but transfer of a thymus-independent immune response to Brucella antigen did not. Cells with surface Ig were involved in both types of adoptive transfers. Thus the presently available T markers do not provide evidence for T cells carrying surface Ig. Suppression of the Ig phenotype by antibody: antigenic modulation? A phenotypic change from Ig⁺ to Ig^- occurs when Ig⁺ lymphocytes or myeloma cells are incubated with anti-Ig sera in vitro in the absence of complement (C). As with antigenic modulation in the TL system, which it resembles, this phenomenon is temperature dependent and in the case of lymph node cells (LNC) can be inhibited by high doses of actinomycin D.     

FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation

FcγRIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell (PC) differentiation. Here, we analyzed the effect of B cell–intrinsic FcγRIIB expression on B cell activation and PC differentiation. Loss of FcγRIIB on B cells in Fcgr2b–conditional KO (Fcgr2b-cKO) mice led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) B cells had the highest expression of FcγRIIB in both mice and humans. This high expression of FcγRIIB was linked to increased MZ B cell activation, Erk phosphorylation, and calcium flux in the absence of FcγRIIB triggering. We observed a marked increase in IgG3⁺ PCs and B cells during extrafollicular PC responses in Fcgr2b-cKO mice. The increased IgG3 response following immunization of Fcgr2b-cKO mice was lost in MZ-deficient Notch2 Fcgr2b–double KO mice. Importantly, patients with systemic lupus erythematosus (SLE) had a decrease in FcγRIIB expression that was strongest in MZ B cells. Thus, we present a model in which high FcγRIIB expression in MZ B cells prevented their hyperactivation and ensuing autoimmunity.     

Genetic control of immune responses in vitro. I. Development of primary and secondary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro

In vivo, the antibody response in mice to the random terpolymer L-glutamic acid⁵⁰-L-alanine³⁰-L-tyrosine¹⁰ (GAT) is controlled by a histocompatibility-linked immune response gene(s). We have studied antibody responses by spleen cells from responder and nonresponder mice to GAT and GAT complexed to methylated bovine serum albumin (GAT-MBSA) in vitro. Cells producing antibodies specific for GAT were enumerated in a modified Jerne plaque assay using GAT coupled to sheep erythrocytes as indicator cells. Soluble GAT stimulated development of IgG GAT-specific plaque-forming cell (PFC) responses in cultures of spleen cells from responder mice, C57Bl/6 (H-2^b), F₁ (C57 x SJL) (H-2^b/s), and A/J (H-2^a). Soluble GAT did not stimulate development of GAT-specific PFC responses in cultures of spleen cells from nonresponder mice, SJL (H-2^s), B10.S (H-2^s), and A.SW (H-2^s). GAT-MBSA stimulated development of IgG GAT-specific PFC responses in cultures of spleen cells from both responder and nonresponder strains of mice. These data correlate precisely with data obtained by measuring the in vivo responses of responder and nonresponder strains of mice to GAT and GAT-MBSA by serological techniques. Therefore, this in vitro system can effectively be used as a model to study the cellular events regulated by histocompatibility-linked immune response genes.     

Studies on human antibodies. 8. Properties and association constants of human antibodies to blood group A substance purified with insoluble specific adsorbents and fractionally fluted with mono- and oligosaccharide

Human antibodies to blood group A substance were purified by absorption on columns of insoluble polyleucyl hog blood group A + H substance and eluted first with N-acetylgalactosamine and then with an A active reduced pentasaccharide AR_L0.52. The γM and γG antibodies in these eluates were separated by density gradient centrifugation. The antibodies were studied for their relative capacities to be inhibited by various blood group A active oligosaccharides. Antibodies eluted by the N-acetylgalactosamine could be inhibited by N-acetylgalactosamine, as well as by lower concentrations of A active tri- and pentasaccharides, while those eluted by the pentasaccharide AR_L0.52 could only be inhibited by the two oligosaccharides, but not by N-acetylgalactosamine, indicating that the N-acetylgalactosamine eluate had more antibodies with smaller size combining sites than the AR_L0.52 eluate. Measurements by equilibrium dialysis gave values ranging from 2 x 10³ to 1 x 10⁵ M ^–1 and the values obtained with the AR_L0.52 eluate were somewhat higher than those with the GalNAc eluate. Only one of three anti-A sera had γM anti-A in the AR_L0.52 eluate, while all three had γM in the N-acetylgalactosamine eluate. Data on the precipitating, hemagglutinating, complement fixing, hemolytic properties of the eluted antibodies, and of their content of κ and λ light chains are given.     

NVX-CoV2373 vaccination induces functional SARS-CoV-2–specific CD4+ and CD8+ T cell responses

NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated individuals made CD4⁺ T cell responses after 1 and 2 doses of NVX-CoV2373, and a subset of individuals made CD8⁺ T cell responses. Characterization of the vaccine-elicited CD8⁺ T cells demonstrated IFN-γ production. Characterization of the vaccine-elicited CD4⁺ T cells revealed both circulating T follicular helper (cTfh) cells and Th1 cells (IFN-γ⁺, TNF-α⁺, and IL-2⁺) were detectable within 7 days of the primary immunization. Spike-specific CD4⁺ T cells were correlated with the magnitude of the later SARS-CoV-2–neutralizing antibody titers, indicating that robust generation of CD4⁺ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 that utilizes Matrix-M adjuvant.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-gamma production

To investigate if transporter associated with antigen processing (TAP)–1 is required for CD8⁺ T cell–mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1^−/−, CD8^−/−, and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1^−/− mice displayed greater susceptibility than CD8^−/−, β₂-microglobulin^−/− (β₂m^−/−), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1^−/− mice correlated with a reduction in the frequency of activated (CD62L^low CD44^hi) and interferon (IFN)-γ–producing CD4⁺ T cells. Interestingly, infected TAP-1^−/− mice also showed reduced numbers of IFN-γ–producing natural killer (NK) cells relative to WT, CD8^−/−, or β₂m^−/− mice, and after NK cell depletion both CD8^−/− and WT mice succumbed to infection with the same kinetics as TAP-1^−/− animals and displayed impaired CD4⁺ T cell IFN-γ responses. Moreover, adoptive transfer of NK cells obtained from IFN-γ^+/+, but not IFN-γ^−/−, animals restored the CD4⁺ T cell response of infected TAP-1^−/− mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-γ–producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.     

α1G-dependent T-type Ca2+ current antagonizes cardiac hypertrophy through a NOS3-dependent mechanism in mice

In noncontractile cells, increases in intracellular Ca²⁺ concentration serve as a second messenger to signal proliferation, differentiation, metabolism, motility, and cell death. Many of these Ca²⁺-dependent regulatory processes operate in cardiomyocytes, although it remains unclear how Ca²⁺ serves as a second messenger given the high Ca²⁺ concentrations that control contraction. T-type Ca²⁺ channels are reexpressed in adult ventricular myocytes during pathologic hypertrophy, although their physiologic function remains unknown. Here we generated cardiac-specific transgenic mice with inducible expression of α1G, which generates Ca_v3.1 current, to investigate whether this type of Ca²⁺ influx mechanism regulates the cardiac hypertrophic response. Unexpectedly, α1G transgenic mice showed no cardiac pathology despite large increases in Ca²⁺ influx, and they were even partially resistant to pressure overload–, isoproterenol-, and exercise-induced cardiac hypertrophy. Conversely, α1G^–/– mice displayed enhanced hypertrophic responses following pressure overload or isoproterenol infusion. Enhanced hypertrophy and disease in α1G^–/– mice was rescued with the α1G transgene, demonstrating a myocyte-autonomous requirement of α1G for protection. Mechanistically, α1G interacted with NOS3, which augmented cGMP-dependent protein kinase type I activity in α1G transgenic hearts after pressure overload. Further, the anti-hypertrophic effect of α1G overexpression was abrogated by a NOS3 inhibitor and by crossing the mice onto the Nos3^–/– background. Thus, cardiac α1G reexpression and its associated pool of T-type Ca²⁺ antagonize cardiac hypertrophy through a NOS3-dependent signaling mechanism.     

Functional heterogeneity of murine lymphoid cells. V. Lymphocytes lacking detectable surface theta or immunoglobulin determinants

An appreciable percent (3–14%) of the lymphocyte-like cells of the mouse spleen lack both the θ-isoantigen and sufficient surface immunoglobulin to be detected by conventional immunofluorescence or autoradiographic procedures. These θ^-,Ig^- cells are increased in frequency after treatment of mice with antithymocyte serum or in mice that have been thymectomized, irradiated (850 R), and reconstituted with bone marrow cells. Moreover, in chimeric C57BL/6 mice in which the T cells are derived from (BALB/c x C57BL/6)F₁ donors, θ^-,Ig^- cells also lack BALB/c histocompatibility antigens. These experiments indicate that θ^-,Ig^- cells are not θ^- T lymphocytes. Removal of complement receptor lymphocytes from spleen cell populations increases the frequency of θ^-,Ig^- cells, indicating that such cells lack the complement receptor. Partially purified populations of θ^-,Ig^- cells have been obtained by cytolysis by anti-θ- and anti-κ-antibody and complement and by density gradient ultracentrifugation. These cells closely resemble lymphocytes in morphology. The only exceptional feature is the existence of prominent nucleoli. The θ^-,Ig^- cells lack hemoglobin and endogenous peroxidases, are not actively phagocytic, and do not adhere to glass. This suggests they are not of the erythroid, myeloid, or monocytoid lines. [³H]Thymidine labeling studies indicate that θ^-,Ig^- cells are members of a relatively slowly dividing cell pool. Whether θ^-,Ig^- cells are members of the "classical" B lymphocyte line or belong to another, as yet undescribed, lineage is not yet certain.     

Nonredundant Roles of IL-10 and TGF-�� in Suppression of Immune Responses to Hepatic AAV-Factor IX Gene Transfer

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-β blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8⁺ T-cell infiltration of the liver. Furthermore, TGF-β was critical for tipping the balance toward an regulatory immune response. TGF-β was required for CD4⁺CD25⁺FoxP3⁺ Treg induction, which was necessary for suppression of effector CD4⁺ and CD8⁺ T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-β in prevention of immune responses against AAV-F.IX-transduced hepatocytes.     

Antigen-specific cells in mouse bone marrow. II. Fluctuation of the number and potential of immunocyte precursors after immunization

Quantitative and qualitative changes of mouse bone marrow cells were studied by limiting dilution assays 2–3.5 months after immunization of donors with sheep erythrocytes or unrelated antigens (Salmonella typhimurium, horse and chicken erythrocytes). Irradiated (C3H x C57BL/10)F₁ mice were reconstituted with an excess of nonprimed thymocytes and small graded numbers of primed bone marrow cells. Direct and indirect plaque-forming cells (PFC) were induced by secondary stimulation with SRBC and enumerated on the 9th day after cell transplantation. Marrow precursors of PFC (P-PFC) cooperated with thymocytes in the production of direct and indirect PFC after SRBC priming. The limiting dilution plots, which were not compatible with predictions of the Poisson model before immunization, changed and conformed to this model afterwards, as if the population of P-PFC had become functionally more homogeneous. The concentration of marrow P-PFC increased up to the 3rd month after priming, and decreased during the 4th, varying over two logarithms of nucleated marrow cells. The fluctuation was simultaneous and of the same order of magnitude for precursors of direct and indirect PFC, which were class restricted. A third effect of immunization was detected at 3.5 months: individual precursor units generated 3–4 times more direct and indirect PFC than at earlier intervals. Qualitative and quantitative changes of marrow P-PFC participating in anti-sheep responses were specific, since antigens unrelated to SRBC failed to induce them. The data suggested that marrow-derived cells were the major carriers of immunologic memory, but that they functioned in cooperation with thymus-derived inducer cells during secondary anti-sheep responses.     

Antigen-specific cells in mouse bone marrow. I. Lasting effects of priming on immunocyte production by transferred marrow

Graded numbers of marrow cells and 5 x 10⁷ thymocytes were mixed in vitro and transplanted into X-irradiated (C3H x C57BL/10)F₁ mice. Upon injection of sheep or chicken erythrocytes, splenic plaque-forming cells secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. Anti-sheep and anti-chicken primary PFC responses elicited by nonimmune marrow cells differed sharply from each other under the conditions of limiting dilution assays. The frequencies of anti-chicken responses in recipients of different numbers of marrow cells conformed to the predictions of the Poisson model, while the frequencies of anti-sheep responses did not. Hence, the function of certain marrow-derived cells was expressed differentially during the two immune responses, to exclude that the same precursor units generated anti-sheep or anti-chicken PFC. The former precursor cells or units were functionally more heterogeneous than the latter. Immunization of marrow donors against sheep erythrocytes did not alter the population of cells engaged in anti-chicken responses, since limiting dilution assays with immune and nonimmune marrow cells gave identical results. However, anti-sheep immunization altered specifically the cell population engaged in anti-sheep responses, in two ways: (a) potentially immunocompetent marrow cells underwent antigen-dependent differentiation or maturation, to become functionally homogeneous. Consequently, the frequencies of PFC responses in limiting dilution assays conformed to the Poisson model; the changes occurred independently in class-restricted precursors of direct and indirect PFC. (b) marrow cells capable of inhibiting precursors of direct anti-sheep PFC arose in primed mice. The inhibition, which was specific, could have been effected directly by marrow cells or by a diffusable product such as IgG antibody. Results indicated that potentially immunocompetent cells of mouse marrow with distinct functions were antigen specific and antigen sensitive.